摘要
为了获得可初步鉴别诊断疫苗免疫和自然感染的蓝舌病病毒(BTV)抗原,试验将8型BTV S10基因中NS3蛋白对应的开放阅读框(ORF)克隆到原核低温诱导表达载体pColdⅠ中,构建重组表达质粒pCold-NS3,然后转化至大肠杆菌BL21(DE3)感受态细胞中,通过对IPTG浓度和诱导温度条件优化,确立重组蛋白NS3最佳表达条件,通过镍柱纯化后用Western-blot和间接ELISA鉴定其免疫活性。结果表明:成功构建了pCold-NS3重组质粒,重组菌株在温度为15℃、以1.0 mmol/L IPTG诱导24 h时重组NS3蛋白表达量最高,分子质量约为29 ku;纯化后的重组蛋白能与BTV自然感染阳性血清和His-tag单克隆抗体发生特异性结合。说明成功获得了具有良好反应原性的BTV重组NS3蛋白。
In order to obtain the preliminary differential diagnosis Bluetongue virus(BTV)antigen to distinguish between natural infection and vaccine immunity,S10 gene of BTV serotype 8 was inserted into the prokaryotic expression vector pColdⅠto obtain the recombinant plasmid pCold-NS3 and then transformed the plasmid into E.coli BL21(DE3).The optimal expression condition of recombinant protein NS3 was determined by optimizing IPTG concentration and induction temperature.The recombinant protein was purified by nickel column and identified by Western-blot and ELISA assay.The results showed that pCold-NS3 recombinant plasmid was successfully constructed.The recombinant strain showed the molecular weight was about 29 ku and the highest expression of the recombinant NS3 protein was obtained when the recombinant strain was induced with 1.0 mmol/L IPTG at 15℃for 24 h.The purified recombinant protein could specifically bind to BTV naturally infected serum and His-tag monoclonal antibody.It indicated that BTV recombinant NS3 protein with good reactivity was successfully obtained.
作者
史卫军
黄超华
曹琛福
杨俊兴
林彦星
王潇
花群义
SHI Weijun;HUANG Chaohua;CAO Chenfu;YANG Junxing;LIN Yanxing;WANG Xiao;HUA Qunyi(Inspection and Quarantine Center for Animals and Plants of Shenzhen Customs,Shenzhen 518045,China;College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2020年第6期74-79,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
国家“十三五”重点研发课题项目(2017YFD0502304)。
关键词
蓝舌病病毒
NS3蛋白
载体构建
原核表达
鉴定
Bluetongue virus(BTV)
NS3 protein
vector construction
prokaryotic expression
identification
作者简介
史卫军(1978-),男,兽医师,本科,研究方向为动物疫病检测,swj10-322@163.com;通信作者:花群义(1965-),男,研究员,博士,研究方向为动物疫病检测,13922861650@163.com.
